Lo sviluppo di questo protocollo è stato finanziato dal California Institute for Regenerative Medicine (CIRM, TR1-01246) e Parkinson Alliance.

La pelle biopsia ottenuta utilizzando procedura standard 3 (ad esempio ottenuto con 4 millimetri strumento Visipunch rotondo) dovrebbe essere tenuto in completo DMEM 20% FBS media su ghiaccio. Una volta che il campione è arrivato in laboratorio, elaborare la biopsia appena possibile. 1. Preparazione della pelle Punch Biopsia PASSI 1,1-1,3 DEVONO essere effettuata in un BIOSAFETY ARMADIO <ol><li> Preparare in anticipo: Aggiungere 1 ml di 0,1% di gelatina a ciascun pozzetto di una piastra da 6 pozzetti. Impostare la piastra da parte per 30-60 min. Aspirare la soluzione di gelatina e aggiungere 800 ml di completo DMEM/20% FBS media per ogni bene. Garantire che l&#39;intera superficie del pozzetto è coperto con supporti. </li><li> Invertire il coperchio di un 10 centimetri piatto di coltura tissutale sterili e aggiungere 1,5 ml di DMEM / FBS 20% media al centro del coperchio e diffondere la media di goccia con la punta della pipetta sierologica. </li><li> Utilizzando una pinza sterile, posto tegli pelle biopsia pezzo nei media sul piatto. </li></ol> 2. Dissezione della pelle Punch Biopsia PASSI 9 2,1-2,2 DEVONO essere effettuata in un ORIZZONTALE cappa a flusso laminare <ol><li> Posizionare la porzione di fondo di 10 cm di piatto su coperchio invertita, e trasferire la biopsia al microscopio da dissezione nella cappa a flusso laminare. </li><li> Sezionare una biopsia della pelle tondo 4 mm in 12-15 pezzi di dimensioni uniformi con bordi taglienti per tagliare pezzi in due metà uguali usando un bisturi per tenere la biopsia a posto e la seconda bisturi per tagliare con un moto di rollio in una direzione. Pezzi con bordi frastagliati contribuiscono alla scarsa attaccamento / cella escrescenza. </li></ol> 3. Trasferimento di pelle Dissected Biopsia Pezzi nel tessuto piastre di coltura PASSI 3,1-3,6 DEVONO essere effettuata in un BIOSAFETY ARMADIO <ol><li> Posizionare la parte inferiore del piatto di coltura 10 cm tissutalesulla parte superiore del coperchio invertita, ed il piatto di trasferimento nella all&#39;armadietto biosicurezza. </li><li> Utilizzando una pinza a punta, posto 2-3 biopsia pezzi in ciascun pozzetto della piastra 6-ben preparato contenente 800 ml e non sui pozzi asciutti. Utilizzare maschiatura o scorrevoli movimento per ottenere i pezzi di allegare al fondo del pozzo. Un bisturi è utile per rimuovere eventuali pezzi di biopsia dalle pinze. </li><li> Posizionare la piastra da 6 pozzetti nel 37 ° C incubatore. Monitorare quotidianamente per garantire che vi sia una pellicola di rivestimento supporti fondo del bene per la prima settimana; aggiungere ~ 200 microlitri ogni 2 giorni sostituirà i supporti evaporate. </li><li> Dopo una settimana, aumentare la quantità di supporti a 2 ml di completo DMEM/20% FBS e cambiamento dei media ogni 2-3 giorni. </li><li> Una volta fibroblasti sono confluenti in ciascun pozzetto al punto in cui i fibroblasti stanno raggiungendo i bordi del pozzo, e trypsinize passaggio 6 pozzetti in 2X T75 beute (passaggio 1). I pezzi di tessuto possono essere trasferiti pure. Essi non attaccare ed essere lavato out durante il prossimo cambiamento dei media. </li><li> Una volta che i fibroblasti sono confluenti, trasferirli 3X T175 fiaschi (passaggio 2), congelare in DMEM completo MEDIA Plus 10% DMSO a 1x10 6 cellule / ml per flaconcino. </li></ol> 4. Caratterizzazione dei fibroblasti attraverso Immunostaining <ol><li> Fibroblasti cultura in 8 e diapositive da camera rivestite di gelatina. </li><li> Quando le cellule sono al 80% di confluenza, aspirare il terreno da ogni pozzetto. </li><li> Fissare le cellule in 4% paraformaldeide per dieci minuti a temperatura ambiente. </li><li> Lavare i pozzetti per 3 volte con PBS 1X. </li><li> Permeabilize le cellule con 150 ml di 0,3% Triton X-100 (in PBS) per 5 min a temperatura ambiente. </li><li> Lavare i pozzetti per 3 volte con PBS 1X. </li><li> Aggiungere 200 microlitri soluzione bloccante (PBS + 5% siero di capra normale (Vector, S-1000)) per 1 ora a temperatura ambiente. </li><li> Preparare una diluizione 1:250 di Anti-Serphin-1 (Anti-coniglio, mAB, Sigma, S5950-200 ml) in soluzione di bloccaggio. </li><li> Aspirate la soluzione di saturazione e aggiungere 150 microlitri della diluizione 1:250 di AntiSERPINH-1. Incubare a 1-2 ore a temperatura ambiente, oppure a 4 ° C durante la notte. </li><li> Lavare i pozzetti per 3 volte con PBS 1X. </li><li> Preparare un anticorpo monoclonale di capra anti-coniglio 1:200 (Alexa Fluor capra anti-IgG di coniglio (H + L), Invitrogen, A11008) nella soluzione di blocco. </li><li> Aggiungere 150 ml di 1:200 anticorpo monoclonale di capra anti-coniglio in ciascun pozzetto. Incubare per 1 ora, al buio, a temperatura ambiente. </li><li> Lavare i pozzetti una volta con 1XPBS. </li><li> Aspirare il PBS, e sollevare con cura le camere per rivelare solo la diapositiva. </li><li> Montare il vetrino con 2-3 gocce di mezzo di montaggio contenenti DAPI (Vector, H-1200). </li><li> Analizzare il vetrino sotto un microscopio a fluorescenza. </li></ol>

<ol>
	<li>Nguyen, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress.">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li><li>Mak, S. K., Tewari, D., Tetrud, J. W., Langston, J. W., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+skin+fibroblasts+from+a+Parkinson's+disease+patient+with+an+alpha-synuclein+triplication.">Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.</a> Journal of Parkinson's Disease. 1, 175-183 (2011).</li><li>Punch Zuber, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=biopsy+of+the+skin.">biopsy of the skin.</a> Am. Fam. Physician. 65, 1155-1164 (2002).</li><li>Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+fibroblast+cultures.">Establishment of fibroblast cultures.</a> Curr. Protoc. Cell Biol. Chapter 2, Unit 2, 1 (2001).</li><li>Kuroda, K., Tajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP47+is+a+useful+marker+for+skin+fibroblasts+in+formalin-fixed,+paraffin+embedded+tissue+specimens.">HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin embedded tissue specimens.</a> J. Cutan. Pathol. , 241-246 (2004).</li></ol>

Gli autori non hanno nulla da rivelare.

Con questo protocollo, le culture relativamente pure di fibroblasti cutanei possono essere ottenuti. I fibroblasti hanno peculiari caratteristiche morfologiche, corpi cellulari mandrino-come allungate, rotonde a nuclei delle cellule ovali, ed i fibroblasti crescono allineati e in fasci, quando confluenti. I mezzi di comunicazione supporta la crescita dei fibroblasti, mentre altre popolazioni cellulari ad esempio cheratinociti bisogno supplementi e fattori di crescita, o sono mitoticamente meno attivi, ciò perm...

<table border="1">
<tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica</td>
 <td>S6E</td>
 </tr>
 <tr>
 <td>10 cm Tissue Culture Petri dish</td>
 <td>VWR</td>
 <td>25382-166</td>
 </tr>
 <tr>
 <td>6-well Tissue Culture plate </td>
 <td>VWR</td>
 <td>73520-906</td>
 </tr>
 <tr>
 <td>Stainless steel disposable sterile scalpels (2), blade No#15, Miltex</td>
 <td>VWR</td>
 <td>21909-660</td>
 </tr>
 <tr>
 <td>Sterile pointed-tip forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Conical tubes</td>
 <td>VWR</td>
 <td>21008-940</td>
 </tr>
 <tr>
 <td>2 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-894</td>
 </tr>
 <tr>
 <td>5 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-896</td>
 </tr>
 <tr>
 <td>10 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-898</td>
 </tr>
 <tr>
 <td>Pasteur Pipettes</td>
 <td>VWR</td>
 <td>14672-380</td>
 </tr>
 </tbody>
</table>
Table A. Table of specific reagents and equipments.
 <table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company </td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1X DMEM: High Glucose</td>
 <td>Invitrogen/Gibco</td>
 <td>11960-069</td>
 </tr>
 <tr>
 <td>20% Fetal Bovine Serum</td>
 <td>Invitrogen/Gibco</td>
 <td>26140-079</td>
 </tr>
 <tr>
 <td>1X L-Glutamine</td>
 <td>Invitrogen/Gibco</td>
 <td>25030-164</td>
 </tr>
 <tr>
 <td>1X MEM Non-essential Amino Acids </td>
 <td>Invitrogen/Gibco</td>
 <td>11140-076</td>
 </tr>
 <tr>
 <td>1X Penicillin/Streptomycin</td>
 <td>Invitrogen/Gibco</td>
 <td>15140-163</td>
 </tr>
 </tbody>
 </table>
Table B. Reagents.

skin punch biopsy explant culture for derivation of primary human fibroblasts

Tessuti e linee cellulari derivate da un individuo con malattia sono fonti ideali per studiare fenotipi cellulari correlate alla malattia. Fibroblasti derivati ​​da pazienti in questo protocollo sono stati utilizzati con successo nella derivazione di cellule staminali pluripotenti indotte alla malattia modello 1. Primi passi di questi fibroblasti possono anche essere utilizzati per saggi funzionali a base di cellule per studiare percorsi specifici di malattia, meccanismi 2 e successive approcci di screening di stupefacenti. Il vantaggio del protocollo presentato nel corso procedure enzimatiche sono 1) la riproducibilità della tecnica di piccole quantità di tessuti derivati ​​da pazienti anziani, ad esempio, pazienti affetti da morbo di Parkinson, 2) l&#39;approccio tecnicamente semplice su metodologie più impegnativi utilizzando trattamenti enzimatici, e 3 ) il corrispettivo tempo: questo protocollo richiede 15-20 minuti e può essere eseguita subito dopo la biopsia arrivo. Trattamenti enzimatici possono richiedere fino a 4 ore e hanno i problemi dioverdigestion, riduzione della vitalità cellulare e la successiva collocazione delle cellule quando non gestita correttamente. Questo protocollo descrive la dissezione e la preparazione di una pelle umana 4 mm per biopsia derivazione di una coltura di fibroblasti e ha un tasso di successo molto alto che è importante quando si tratta di campioni di tessuti derivati ​​da pazienti. In questa cultura, cheratinociti migrano fuori dal tessuto biopsia entro la prima settimana dopo la preparazione. I fibroblasti compaiono 7-10 giorni dopo la prima conseguenza di cheratinociti. DMEM High Media glucosio addizionato con 20% FBS favorisce la crescita dei fibroblasti su cheratinociti e fibroblasti sarà invadere i cheratinociti. Dopo 2 passaggi cheratinociti sono stati diluiti in colture di fibroblasti con conseguente relativamente omogenei che esprime la fibroblasti marcatore SERPINH1 (HSP-47). Usando questo approccio, 15-20 milioni fibroblasti possono essere derivate in 4-8 settimane per la raccolta di cellule. La dissezione pelle dura circa 15-20 minuti, le cellule vengono poi monitorati una volta al giornosotto il microscopio, e dei media è cambiata ogni 2-3 giorni dopo il sequestro e la conseguenza di cellule.

Cheratinociti crescere senza i pezzi bioptici appena 48 ore dopo la dissezione. Prima fibroblasti escrescenza può essere osservato circa una settimana dopo l&#39;elaborazione. Una volta che i pozzi hanno raggiunto confluenza, i fibroblasti sono diversi passaggi per altri due passaggi per raggiungere tre 150 centimetri palloni (T150) e le cellule vengono crioconservati. Noi generiamo con questo metodo di 15-20 milioni cellule. I fibroblasti sono positivi per anti-SERPINH1 (noto anche come HSP-47) (Figura 1),</st...

Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Il protocollo cultura espianto di fibroblasti da biopsie cutanee umane è un modo tecnicamente robusto e semplice per ricavare cellule della pelle all&#39;interno di 4-8 settimane per il settore bancario di circa 15-20 milioni di cellule in un numero basso passaggio.

Pelle Punch Biopsia Espianto Cultura per la derivazione di fibroblasti umani primari

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

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40.8K Views.  The Parkinson's Institute. Il protocollo cultura espianto di fibroblasti da biopsie cutanee umane è un modo tecnicamente robusto e semplice per ricavare cellule della pelle all'interno di 4-8 settimane per il settore bancario di circa 15-20 milioni di cellule in un numero basso passaggio.

Video: Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Generazione sperimentale di carcinoma-Associated fibroblasti (CAF) da fibroblasti umani mammari

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

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Medicina

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

L&#39;uso di fibroblasti umani primari per il monitoraggio fenotipi mitocondriali nel campo della malattia di Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In vitro organoide Cultura di primari tumori del colon mouse

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Three-dimensional Tissue Culture modello per studiare Primario Osso Osseo e le sue Malignità

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Cultura primario di Human vestibolare Schwannomi

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Scheletrici Procedure biopsia muscolare dell&#39;uomo con la tecnica Bergström Modificato

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Istituzione di umano epiteliali Enteroids e Colonoids dal tessuto intero e biopsia

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Umano<em&gt; vasto laterale</em&gt; Biopsia muscolare scheletrico Utilizzando la Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Tissue Caratterizzazione dopo un metodo nuovo disaggregazione per Skin Micro-innesti Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualizzazione quantitativa di leucociti infiltrati in un modello di murina della miocardite fulminante mediante microscopia a foglio chiaro

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...